Dokanehiifard Sadat, Soltani Bahram M
Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
Gene. 2018 Jan 30;641:297-302. doi: 10.1016/j.gene.2017.10.075. Epub 2017 Oct 27.
Wnt signaling plays important roles in differentiation, morphogenesis and development. This signaling pathway is highly regulated at all levels and microRNAs are small noncoding RNAs regulating Wnt signaling. Here, we intended to investigate hsa-miR-11181 (a novel miRNA located in TrkC gene) effect on Wnt signaling pathway in SW480 cell line. TOP/FOP flash assay indicated up-regulation of Wnt signaling, following the overexpression of hsa-miR-11181, verified through RT-qPCR. Bioinformatics analysis predicted APC1, APC2 and Axin1 might be targeted by hsa-miR-11181. Then, RT-qPCR analysis indicated that APC2 and Axin1 have been significantly down-regulated following the hsa-miR-11181 overexpression. However dual luciferase assay analysis supported only APC2 3'-UTR is directly targeted by this miRNA. Then, treatment of SW480 cells with Wnt-inhibitory small molecules supported the effect of hsa-miR-11181 at the inhibitory complex level containing APC2 protein. Consistently, viability of SW480 cells overexpressing hsa-miR-11181 was significantly elevated, measured through MTT assay. Overall, these results suggest that hsa-miR-11181 may play a crucial role in Wnt signaling regulation and confirmed that APC2 3'-UTR is targeted by hsa-miR-11181 and propose the presence of its recognition sites in the promoter or coding regions of Axin1 gene.
Wnt信号通路在细胞分化、形态发生和发育过程中发挥着重要作用。该信号通路在各个层面均受到高度调控,而微小RNA是一类可调控Wnt信号通路的小型非编码RNA。在此,我们旨在研究hsa-miR-11181(一种位于TrkC基因中的新型微小RNA)对SW480细胞系中Wnt信号通路的影响。TOP/FOP荧光素酶报告基因检测表明,hsa-miR-11181过表达后Wnt信号通路上调,这一结果通过逆转录定量聚合酶链反应(RT-qPCR)得以验证。生物信息学分析预测,APC1、APC2和Axin1可能是hsa-miR-11181的靶标。随后,RT-qPCR分析表明,hsa-miR-11181过表达后,APC2和Axin1显著下调。然而,双荧光素酶检测分析表明,只有APC2的3'-非翻译区(3'-UTR)是该微小RNA的直接靶标。然后,用Wnt抑制性小分子处理SW480细胞,证实了hsa-miR-11181在含有APC2蛋白的抑制复合物水平上的作用。同样,通过MTT检测发现,过表达hsa-miR-11181的SW480细胞活力显著升高。总体而言,这些结果表明,hsa-miR-11181可能在Wnt信号通路调控中发挥关键作用,并证实APC2的3'-UTR是hsa-miR-11181的靶标,且提示在Axin1基因的启动子或编码区存在其识别位点。